Supercritical fluid chromatography (SFC) is a separation technique similar to high performance liquid chromatography (HPLC), except one of the fluids used as a solvent is a highly compressible liquefied gas. Supercritical fluid extraction (SFE) is a related technique but with somewhat lower requirements for accurate flow or pressure control. The most common fluid used in SFC (and SFE) is carbon dioxide (CO2), which will be considered as representative of all such fluids.
At room temperature and atmospheric pressure, carbon dioxide is a low density gas (density approximately 0.002 g/cm3). The desirable characteristics of carbon dioxide for SFC and SFE are only achieved when the carbon dioxide is held at a liquid-like density, usually between 0.6 and 1.0 g-cm-3, by raising its pressure to 80 to 600 Bar, while keeping the temperature in the general range of 20° to 100° C., and more commonly between 35 to 60° C. Under such conditions, the carbon dioxide: 1) acts as a solvent, 2) exhibits very high solute binary diffusion coefficients (allows higher flow rates than in HPLC), and 3) exhibits very low viscosity (generates lower pressure drops across columns compared to HPLC).
To be useful in SFC (or SFE), the carbon dioxide is compressed to high pressures and pumped as a liquid or as a supercritical fluid, at a liquid like density, through a separation column. To prevent it from expanding to atmospheric pressure in the column, a back pressure regulator (BPR) is typically placed downstream to keep the column outlet pressure typically above 80 Bar. Detectors capable of operating under high pressure may be mounted between the column and the BPR. Low pressure detectors may be mounted in the flow stream directly downstream of the BPR.
Several problems exist with the recovery of purified liquid fractions from gas-liquid separators used in preparative supercritical fluid chromatography [SFC] systems. Preparative SFC uses near-critical temperature carbon dioxide [C02] at supercritical pressures [>72.9 bar] as one major constituent of the mobile phase and liquid organic modifiers as the other. At high pressures, these components as well as high levels of dissolved solids requiring purification are soluble and/or miscible into a single fluidic phase. The combined mobile phase entrained with aliquots of dissolved sample is passed through a separation column from which purified flow segments of the sample called “peaks” emerge. It is the goal of the preparative SFC system to direct individual peaks either to clean, empty containers or to containers filled previously with peaks containing the same solute from prior injections—a process known as “pooling.”
At the end of the Prep SFC chromatographic process, peaks pass through a back pressure regulator which relieves the fluid of the high pressure required for separation. This rapid pressure reduction causes an immediate expansion of the C02 component to a gas—a process which is highly endothermic. Hence, the original mobile phase of the flow line carried away from the back pressure regulator becomes biphasic with a very cold mixture of C02 and modifier vapor and liquid modifier containing the dissolved solutes of the peak.
The C02 evaporation process following the backpressure regulator is a very turbulent and chaotic process where the original mobile phase is transformed into a rapidly expanding vapor cloud containing very small droplets, or aerosols, of original liquid modifier saturated with C02. The flow rate of the vapor is limited by the kinematic viscosity of the particular gasses that comprise it—in this case C02 with a small amount of organic vapor. Because of the inherent pressure differential between the exit of the BPR and the downstream path, the vapor continues expanding not just due to the phase change, but due to the pressure differential of the flow line constraining it. Thus, the C02 vapor velocity actually accelerates as it moves down the flow line.
In current commercial Prep SFC systems with flows exceeding 5-10 mL/min, the expanding flow stream is generally first passed through a heater then on to one or more gas liquid separators which vent the vast majority of vapor out a vent port while retaining or transferring the liquid component containing solute to an appropriate storage container.
The original gas-liquid separators for Prep SFC were high pressure cyclonic separators that maintained an internal pressure of five or more bar to limit the expansion of the C02 during the liquid separation process. High velocity streams of the vapor/aerosol mixture were introduced tangentially to a cylindrical separator, which typically had a tapered bottom and an axial vent extending through the top. The flow would continuously be redirected by the separator wall to develop a circular flow pattern. The dense components would collect near the wall due to higher momentum forces. As a result, the heavier aerosols would build up to form a film on the container wall and fall to the bottom under forces of gravity, while the vapor components would find the central path to the vent of least resistance.
Cyclonic separators were not perfect, and a percentage of the original aerosol, containing valuable analytes held in suspension, typically found a path to the vent as well, causing loss of sample and possible contamination of downstream components. Further, the separators were expensive and had to be built for a specific capacity (typically less than one-third of the container volume) to allow sufficient room for expansion of the entering vapor cloud. Once the limited liquid capacity of the separator was reached, the container had to be emptied. One means was by opening a valve which released the C02-saturated liquid to an external container, which caused a violent outgassing and potential rupture of the container. A second means was to depressurize the container, then empty it, which was inefficient and time consuming. Finally, because the collection process deposits thin films of solute-laden liquid on the walls, each separator must be thoroughly cleaned (typically manually) before re-use with a different collection peak, which again was inefficient and time-consuming.
In prior cyclonic separators for each injection, a separate separator was used to collect an individual peak. The cyclones were typically connected in series with three way-valves either diverting to the collector or passing the flow onto the next collector. A final cyclone in the series collected the waste stream. Such systems were most frequently used isocratically (i.e. no change in the modifier composition during a separation) with a series of repeat injections of the same sample. This pooling process is used to purify larger quantities of material than can be well separated by a single injection. The serial nature of collection through cyclonic separators can introduce an additional difficulty. As fractions are collected first by one cyclone and then the next, the delay period between detection and collector shifts. This time shift is a function of tubing size and flow rate and must be accounted for in accurate fraction collection. Missing the time window by as much as a few seconds can cause contamination of the previously pooled fractions and require repurification.
Another prior preparatory SFC fraction collector with open bed fraction collection uses a low backpressure gas-liquid separator which drains continuously at a rate slightly greater than liquid is added. Exiting modifier is directed to a delivery probe via a transfer line at the bottom of the separator. Because the separator is continuously drained, it can be relatively small and easily rinsed. Since it does not accumulate liquid, the next peak entering the separator, even at relatively close timing, should be relatively immune to mixing with the immediately prior peak. As a result, this system has the decided advantage that a single gas liquid separator can handle all fractions from a given injection. The individual fractions are simply diverted to separate containers in the robotic bed.
Timing is a critical element of the success of this system. From the time a peak is detected and or confirmed to the time it will reach the probe divert valve is a critical value. In addition, there is a strong desire not to cause the liquid of the peak to become overly dilute or “broadened” after the detector to the point where it cannot be seen. Factors that can vary this timing can dramatically interfere with both recovery and purity of collected fractions.
One such factor, the drain rate, is controlled by a combination of backpressure, modifier viscosity, and transfer line flow resistance and so must be carefully implemented for a given system. Since the goal is to completely remove entering liquid without accumulation in the splitter, a small amount of the vapor is inevitably entrained in the transfer line as well. For any given isocratic separation, the conditions can be relatively easily adjusted to give reasonable results. However, a problem arises for gradient separations which constitute the primary intended use of the system. Gradient separations are those that systematically alter the mobile phase composition or flow rate during the course of a single injection. A linear modifier gradient changes the composition of modifier continuously over the course of a separation. A step gradient may hold the composition constant for a time them abruptly change the composition. A flow gradient may simply vary the total or modifier flow rate according to some programmed profile.
Varying modifier and/or total flow in the system introduces the following problem. The system conditions must be set to accommodate the highest level of modifier flow at the lowest pressure condition. As a result, the transfer line must be less restrictive than it ordinarily might be for conditions of low modifier at the beginning of a gradient. The unrestrictive line will pass a significant amount of vapor under these conditions and potentially cause difficulty at the probe due to spraying. In addition, the timing of peaks being delivered to the probe becomes much more variable as more gas in entrained at constant pressure. This is because the gas pushes the liquid at indeterminate speeds through the transfer line as a function of pressure drop along the line. Thus it is very difficult to time the start and stop of collection at the probe tip. The problem becomes much worse as the user varies modifier viscosity. At low viscosities such as methanol (0.3 cp) a fairly restrictive tube still carries a significant flow at moderate pressure. As a result, the flow of vapor through the tube is rather limited. If, however, the modifier is changed to n-butanol (cp>2.9 cp) the tube must be made 10-fold less restrictive for the same flow range. Now at least ten times more vapor is released to the probe and can cause serious problems with timing, dispensing and aerosol formation.
Prior systems have taken some unusual steps in an attempt to overcome these problems. First, a makeup pump is added to the system for the purpose of delivering a fixed rate of modifier to the separator. The makeup pump is programmed to a reverse flow gradient, initially delivering a high flow rate and tapering if a smaller rate as the gradient delivers more modifier. Thus, a maximum amount of modifier most be collected at all times by the system. Second, users are advised to use only a single modifier viscosity to avoid having to change flow lines. The result is a significantly less flexible system than desired with unnecessarily large fractions collected due to the makeup flow.
A solution is therefore needed for both high and low pressure gas-liquid separators to provide substantially better deterministic draining to remote containers.